As detailed in the Request For Applications to which this proposal responds, the mechanisms of resistance of some individuals to HIV-1 infection (highly exposed seronegative, HESN) remain poorly understood. Major questions regarding this phenomenon were raised at an NIH workshop on this topic that serves as the basis for this RFA, including: 1) What is different in HESN versus those who get infected? 2) What is the immune response in HESN and is it just a marker of exposure or a correlate of protection? 3) What are the HESN host factors that help HESN resist infection? This proposal addresses these issues by exploring the overarching hypothesis that innate immune reactivity is a key determinant of CD4+ T lymphocyte activation at mucosal surfaces, and therefore a key determinant of susceptibility to HIV-1 infection of an individual. HESN may be different in terms of innate immune reactivity, which would be a key difference in the immune response of these persons compared to the general population, and not necessarily HIV-1-specific. The host factors determining this difference likely would be genetic, which will be explored by whole genome single nucleotide polymorphism examination in a complementary study. We will pursue this hypothesis by examining a unique cohort of men in the Multicenter AIDS Cohort Study who had extremely high-risk sexual exposures in the early- to mid-1980s, yet remained uninfected. The susceptibility and reactivity of their mucosal innate immune system will be assessed using HIV-1 infection and examining the effects of defined stimuli of pattern recognition receptors. We will focus on their site of sexual exposure, the rectal mucosa. Specifically, we propose: To determine innate immune signals important for HIV-1 replication in gut mucosa and quantitate the ability of gut mucosa from HESN to support HIV-1 replication To assess the innate immune responsiveness of HESN gut mucosa to different stimuli To examine the phenotypes of antigen-presenting cells in the gut mucosa of HESN